It has become clear, that ubiquitin-dependent protein degradation can be thought of as a specialized case of protein localization. Ubiquitin is first adenylated by an activating enzyme (El), then transferred to a conjugating enzyme (E2) as a thiol ester, and finally attached to a target protein by ligase (E3). The linkage formed is an isopeptide bond between the carboxyl-terminus of ubiquitin and a side chain lysine on the target protein. The initial conjugation of ubiquitin to a target protein can be followed by conjugation of a second ubiquitin to a lysine residue of the first, and subsequent elongation of this polyubiquitin chain. It is aK481inked polyubiquitin chain that targets the protein for localization to the proteasome and subsequent proteolysis. Polyubiquitin can also exist in several different forms involved in a variety of proteolytic and non-proteolytic processes. Linkages between G76 on one ubiquitin and K48 on the next have been shown to target proteins to the proteasome. Linkages to K63 are involved in receptor endocytosis and sorting, repair of DNA damage, signaling through the TRAF pathway of NFkappaB induction, and the stress response. These are thought to be non-proteasomal functions. Linkages to K29 have been shown to be required for the degradation of proteins bearing a non-hydrolyzable ubiquitin at the N-terminus. Finally, K11-1inked ubiquitin is formed by the CHIP ubiquitin ligase acting on the chaperone BAG-1. Clearly, the diversity of possible linkages and their association with very different physiological processes suggests the presence of specific ending proteins that can recognize and target these alternatively linked chains. The major question to be addressed in this proposal is how these different chains are recognized by polyubiquitin binding proteins. Specific Aim 1 will characterize the binding of polyubiquitin to Isopeptidase T. Specific Aim 2 will determine the substrate specificity and mechanism of Ubp6/Uspl4. Specific Aim 3 will determine if UCH-L1can act as a ubiquitin ligase and examine the consequences of the S 18Y polymorphism. Specific Aim 4 will identify and characterize linkage-specific polyubiquitin binding proteins.